Search

Surprising excitation-dependent, dual emission from a small organic model fluorophore is reported. Gas-phase fluorescence spectra of proflavine (a diaminoacridine) ions reveal two long-lived emitting states, with distinct bands separated by just 1700 cm

There is a need in the field of biological mass spectrometry for structural tools which can report on regional, rather than solely global, structure of gaseous protein ions. Site-specific hydrogen-deuterium (H/D) exchange has shown promise in fulfilling this need, but requires additional method development to prove its utility. In this study, we use H/D exchange and electron capture dissociation (ECD) to probe the gaseous structure of two peptides which are α-helical in solution and which differ by a single point mutation. Global H/D exchange levels, ECD fragmentation profiles, and region specific H/D exchange profiles are compared between wild type (WT) melittin, which adopts a hinged helix conformation in solution, and a mutant P14A melittin which folds into a single helix in solution. High protection from H/D exchange by both peptides is consistent with retention of secondary structure in the gas phase (or refolding into some other compact structure). The P14A mutant melittin exhibits lower ECD fragmentation efficiency than WT melittin, suggesting that it contains more secondary structure in the gas phase, which may indicate that these peptides retain some memory of their solution-phase structures. Examination of the isotopic distributions of fragment ions derived from H/D exchange with subsequent ECD reveals that the C-terminus of these peptides adopts multiple conformations. The results reported here offer insight into the stability of alpha helices in the gas phase, and also highlight the value of combining gas-phase H/D exchange with electron capture dissociation to interrogate gaseous peptide conformation.

The increased utility of fluorescence-based methods in recent years has highlighted the need for brighter, more efficient fluorophores. In order to design these fluorophores, an improved fundamental understanding is necessary of the structural components that intrinsically effect fluorescence efficiency. Here, we characterize the intrinsic effects of deuteration on fluorescence from gaseous oxazine dyes, without the influence of dye-solvent interactions, by making use of an ion trap mass spectrometer that has been altered to enable optical measurements. Comparison of emission spectra of four oxazine dyes: cresyl violet, oxazine 4, oxazine 170, and darrow red, show little change in profile upon deuteration of amine groups. However, deuteration significantly increases the efficiency of fluorescence with an increase in fluorescence lifetime and brightness by 10-23% for the gaseous dyes. This increase is less than half that of the quantum yield increase observed in deuterated solution. This indicates the large fluorescence efficiency changes for the oxazine dyes in deuterated solution result from a combination of both intrinsic effects as well as substantial contribution from altered fluorophore-solvent interactions. The intrinsic effects behind increased lifetime upon deuteration are explored using time-dependent density functional theory (TD-DFT) calculations of potential energy surfaces (PESs) for ground and low lying excited electronic states. In accord with experimental observations, calculated S1-S0 emission spectra show only minor differences between deuterated and non-deuterated forms indicating that the deuteration does not affect the radiative channel appreciably. Relaxed PES scans along the torsional motions of the amino groups reveal that the increase in lifetimes upon deuteration is likely due to quenching of different radiationless changes channels in different oxazine dyes. Calculations suggest that tunneling to access twisted intramolecular charge transfer states in S1 is critical in several of the oxazines. However, in at least one of the dyes examined, the large isotope effect is more likely due to differences in intersystem crossing rates. Overall, this combined experimental and computational investigation elucidates the photophysics of a well-known fluorescent scaffold and provides insight into how small differences can dramatically affect fluorescence outcomes.

The rational design of rhodamines and other fluorescent probes for different functions would benefit from an improved understanding of their photophysics. Key photophysical properties, including fluorescence, depend on the outcome of competing pathways for intra- and intermolecular energy flow within and from excited state molecules. In the work reported here, we simplify this complex landscape by eliminating solvent interactions, revealing intrinsic photophysical effects of systematic structural changes. Selected-ion laser-induced fluorescence (SILIF) is used to examine the effects of stepwise N-methylation on a rhodamine scaffold, starting with the simple rhodamine 123, in the gas phase. Fluorescence excitation and emission spectra together with fluorescence lifetime measurements are reported and discussed. While the systematic decrease in gas-phase 0-0 transition energy by 500 cm-1 per methylation is in line with expectations from solution studies, other trends are observed that are not apparent in solution studies. These include a notable narrowing of spectral profiles, three-fold decrease in Stokes shift and an ∼three-fold increase in brightness as the number of N-methylations rises from zero to four. Most surprising, while rhodamine 123 displays the expected textbook mirror-image symmetry between excitation and emission spectra, the emission spectrum of its tetra N-methylated derivative is ∼30% broader than the excitation spectrum. The likelihood that this difference reflects emission prior to complete vibrational redistribution of energy within the excited state of the larger rhodamines is discussed. This suggestion goes against conventional wisdom about the timescale of energy redistribution within molecules of this size, an understanding which was developed from solution studies. Overall, this study furthers our understanding of energy flow within an important class of fluorophores, highlights the consequences of energy flow between fluorophores and surrounding solvent, and provides benchmark experimental data for solvent-free chromophores to assist and calibrate computational work.

In nature, the finely tuned photophysical properties of chlorophyll a (Chla) are vital to the capture and transfer of sunlight during photosynthesis. In order to better understand how these properties are influenced by the molecular environment, we have examined the intrinsic spectroscopy of Chla in vacuo. Visible photodissociation action spectra (an indirect measure of absorption) of gaseous protonated Chla and Chla complexed with metal cations are reported. These show that spectral features within the Soret band (∼350-445 nm) have markedly different intensities depending on the identity of the cation. In contrast, fluorescence emission spectra of metalated Chla complexes show only small dependences on the identity of the metal ion, with emission maxima shifting from 661 to 654 nm. Remarkably, replacing the metal ion with a proton turns off the fluorescence of this key pigment. Density functional theory geometry-optimized structures indicate that the most favorable site of protonation differs from that of metal cationization, and may help explain the surprising on/off behavior of Chla's intrinsic fluorescence.

The fluorescence enhancement ("turn-on") response of the amyloid-sensing dye thioflavin T (ThT) is examined in vacuo, where solvent interactions are absent. Upon the complexation of ThT with a derivatized β-cyclodextrin, heptakis-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin, turn-on responses in both the gas phase and solution phase were observed. In contrast, turn-on response was not detected when ThT was bound to gaseous cucurbit[7]uril or human telomeric DNA 22AG, whereas clear turn-on response occurs in solution. The observed difference in turn-on response in the gas phase emphasizes the key interplay between chromophore, host and solvent and demonstrates the utility of gas-phase spectroscopy to tease out the balance among intermolecular forces driving the behavior of important chromophores in solution.

A key utility of fluorophores lies in sensing applications: the detection of changes to emission caused by differences in their microenvironment. The rational design of fluorescent sensors remains a significant challenge because of the complexity of factors which control molecular deactivation pathways. Here, in an effort to define the structural criteria underlying the fluorescence turn-on response of Hoechst 33258 (H33258) upon binding to the DNA minor groove, we examine this sensor's intrinsic properties in minimalist microenvironments. We first characterised the intrinsic photophysics of gaseous mono- and di-protonated H33258 ions, then introduced intermolecular interactions by complexation with double-stranded (ds) DNA. Selected-ion laser-induced fluorescence (SILIF) and photodissociation of the gaseous monoprotomers indicate the presence of multiple populations with distinct fluorescence and dissociation properties. We assign one of these to a kinetically-trapped form which is protonated at the site favored in solution. The other form exhibits a more intense emission band which is shifted by more than 6000 cm-1 to the red of the first form. Quantum chemical calculations reveal that this second population is likely a newly-identified protomer, which is considerably more stable in the gas phase than conformations with the solution protonation site. Two routes that increase the fluorescence of H33258 in solution - formation of the diprotomer and complexation with dsDNA - do not produce an increase in fluorescence in the gas phase. However, two other outcomes parallel behaviour. First, the similarity of action spectra of the gaseous dsDNA-H33258 complex and the unbound diprotomer suggest that the dye may be diprotomeric when in complex with gaseous dsDNA. Second, the photodissociation power dependence measurements indicate the presence of at least two distinct populations of both H33258 in complex with dsDNA and in its unbound diprotomeric form. Overall, the results reported here reveal unexplored aspects of the potential energy landscape of H33258, including a new, stable, highly-fluorescent form that may be useful to consider in sensing applications. Moreover, the results reinforce how structure, deactivation pathways and other photophysical properties are intertwined for this DNA-binding dye, which may offer strategies for improved control of DNA-targeting drugs and sensors.

Current peroxisome proliferator-activated receptor (PPAR)-targeted drugs, such as the PPARγ-directed diabetes drug rosiglitazone, are associated with undesirable side effects due to robust agonist activity in non-target tissues. To find new PPAR ligands with fewer toxic effects, we generated transgenic zebrafish that can be screened in high throughput for new tissue-selective PPAR partial agonists. A structural analog of coenzyme Q10 (idebenone) that elicits spatially restricted partial agonist activity for both PPARα and PPARγ was identified. Coenzyme Q10 was also found to bind and activate both PPARs in a similar fashion, suggesting an endogenous role in relaying the states of mitochondria, peroxisomes and cellular redox to the two receptors. Testing idebenone in a mouse model of type 2 diabetes revealed the ability to reverse fatty liver development. These findings indicate new mechanisms of action for both PPARα and PPARγ, and new potential treatment options for nonalcoholic fatty liver disease (NAFLD) and steatosis. This article has an associated First Person interview with the first author of the paper., Summary: A zebrafish screen identifies a novel PPARα/γ ligand, idebenone, with potential for treatment of fatty liver diseases, as seen by testing it in a mouse model of type 2 diabetes.

Ethidium is a cationic dye with fluorescence that is enhanced ∼9-fold upon binding DNA. In order to better understand how the local environment modulates the behavior of this dye, we measured the photophysical properties of gaseous ethidium ions, using a quadrupole ion trap mass spectrometer that has been modified for fluorescence spectroscopy. The photodissociation maximum of gaseous ethidium measured through action spectroscopy is 485 nm and the emission maximum is 548 nm. The Stokes shift (2370 cm−1) of gaseous ethidium is marginally larger than that of ethidium in non-polar solvents, and significantly less than that in polar solvents. Time-resolved fluorescence measurements of gaseous ethidium ions show two components with lifetimes of 21.4 ± 1.5 and 5.1 ± 0.7 ns, which suggest the presence of multiple conformations in the gas phase. Both lifetimes are significantly longer than that of aqueous ethidium, while the longer of the two lifetimes is remarkably similar to that of ethidium in complex with double-stranded DNA in solution. In line with this, the estimated quantum yield of gaseous ethidium is ∼30% lower than that of ethidium in complex with DNA in solution, and ∼10-fold higher than that of aqueous ethidium. These benchmark results provide a reference from which to better understand the factors that modulate the fluorescence of phenanthridine-based dyes by the local environment.

Photodissociation action spectroscopy is often used as a proxy for measuring gas-phase absorption spectra of ions in a mass spectrometer. Although the potential discrepancy between linear optical and photodissociation spectra is generally acknowledged, direct experimental comparisons are lacking. In this work, we use a quadrupole ion trap that has been modified to enable both photodissociation and laser-induced fluorescence to assess how closely the visible photodissociation action spectrum of a fluorescent dye reflects its fluorescence excitation spectrum. Our results show the photodissociation action spectrum of gaseous rhodamine 110 is both substantially narrower and slightly red-shifted (∼120 cm(-1)) compared to its fluorescence excitation spectrum. Power dependence measurements reveal that the photodissociation of rhodamine 110 requires, on average, the absorption of three photons whereas fluorescence is a single-photon process. These differing power dependences are the key to interpreting the differences in the measured spectra. The experimental results provide much-needed quantification and insight into the differences between action spectra and linear optical spectra, and emphasize the utility of fluorescence excitation spectra to provide a more reliable benchmark for comparison with theory.

Laser-induced fluorescence is used to visualize populations of gaseous ions stored in a quadrupole ion trap (QIT) mass spectrometer. Presented images include the first fluorescence image of molecular ions collected under conditions typically used in mass spectrometry experiments. Under these “normal” mass spectrometry conditions, the radial (r) and axial (z) full-width at half maxima (FWHM) of the detected ion cloud are 615 and 214 μm, respectively, corresponding to ~6 % of r0 and ~3 % of z0 for the QIT used. The effects on the shape and size of the ion cloud caused by varying the pressure of helium bath gas, the number of trapped ions, and the Mathieu parameter qz are visualized and discussed. When a “tickle voltage” is applied to the exit end-cap electrode, as is done in collisionally activated dissociation, a significant elongation in the axial, but not the radial, dimension of the ion cloud is apparent. Finally, using spectroscopically distinguishable fluorophores of two different m/z values, images are presented that illustrate stratification of the ion cloud; ions of lower m/z (higher qz) are located in the center of the trapping region, effectively excluding higher m/z (lower qz) ions, which form a surrounding layer. Fluorescence images such as those presented here provide a useful reference for better understanding the collective behavior of ions in radio frequency (rf) trapping devices and how phenomena such as collisions and space-charge affect ion distribution.

Encapsulation of dyes by cucurbituril macrocycles has proven profitable as a strategy to alter fluorescence characteristics in useful ways. Encapsulation generally results in longer fluorescence lifetimes, enhanced brightness, and solvatochromic effects not normally seen in the condensed phase. These effects have been attributed variously to both the removal of interactions with solvent molecules and to the confined environment of extremely low polarizability provided by the cucurbituril interior. It is difficult to disentangle these effects in solution. Here, we present results from gas-phase experiments designed to separate these effects, using cucurbit[7]uril (CB7), and the cationic dye acridine orange (AOH(+)) as a probe. Fluorescence properties of gaseous AOH(+) are compared with those of the gaseous AOH(+)-CB7 complex and with the properties of the dye and complex in aqueous solution. The dependence on the local environment of several spectroscopic properties is discussed, including the fluorescence excitation and emission maxima, the size of the Stokes shift, fluorescence lifetime and relative brightness. An understanding of the modulation of fluorescence properties by the local environment, such as that promoted by this work, will aid in the rational design of improved fluorophores and fluorescent sensors.

The deoxynojirimycin (DNJ) family of imino sugars are glucose analogues with an NH group replacing the oxygen atom in the pyranose ring. They are powerful inhibitors of glycosidases and ceramide glucosyltransferases. The conformation of N-benzyl-DNJ, isolated in the gas phase, is studied using a combination of resonant two-photon ionization (R2PI), UV/UV hole-burn, and IR ion-dip spectroscopies in conjunction with electronic structure theory calculations. Three distinct conformers, one major and two minor, are present and all three are assigned to structures in which the exocyclic hydroxymethyl group is axial to the piperidine ring (gauche- to the ring nitrogen). This contrasts with the preferred equatorial (gauche+) orientation observed for simple glucosides and may well contribute to the stronger binding of some enzymes to DNJ-based inhibitors compared to their natural glucoside substrates.

An improved understanding of the extent to which native protein structure is retained upon transfer to the gas phase promises to enhance biological mass spectrometry, potentially streamlining workflows and providing fundamental insights into hydration effects. Here, we investigate the gaseous conformation of a model β-hairpin peptide using gas-phase hydrogen-deuterium (H/D) exchange with subsequent electron capture dissociation (ECD). Global gas-phase H/D exchange levels, and residue-specific exchange levels derived from ECD data, are compared among the wild type 16-residue peptide GB1p and several variants. High protection from H/D exchange observed for GB1p, but not for a truncated version, is consistent with the retention of secondary structure of GB1p in the gas phase or its refolding into some other compact structure. Four alanine mutants that destabilize the hairpin in solution show levels of protection similar to that of GB1p, suggesting collapse or (re)folding of these peptides upon transfer to the gas phase. These results offer a starting point from which to understand how a key secondary structural element, the β-hairpin, is affected by transfer to the gas phase. This work also demonstrates the utility of a much-needed addition to the tool set that is currently available for the investigation of the gaseous conformation of biomolecules, which can be employed in the future to better characterize gaseous proteins and protein complexes. Graphical Abstract ᅟ.

Knowledge of the structure of protein-ligand complexes can aid in understanding their roles within complex biological processes. Here we use electrospray ionization (ESI) coupled to a Fourier transform ion cyclotron resonance mass spectrometer to investigate the noncovalent binding of the macrocycle cucurbit[7]uril (CB7) to bovine insulin. Recent condensed-phase experiments (Chinai et al., J. Am. Chem. Soc. 133:8810-8813, 2011) indicate that CB7 binds selectively to the N-terminal phenylalanine of the insulin B-chain. Competition experiments employing ESI mass spectrometry to assess complex formation between CB7 and wild type insulin B-chain vs. a mutant B-chain, confirm that the N-terminal phenylalanine plays in important role in solution-phase binding. However, analysis of fragment ions produced by electron capture dissociation (ECD) of CB7 complexed to intact insulin and to the insulin B-chain suggests a different picture. The apparent gas-phase binding site, as identified by the ECD, lies further along the insulin B-chain. Together, these studies thus indicate that the CB7 ligand migrates in the ESI mass spectrometry analysis. Migration is likely aided by the presence of additional interactions between CB7 and the insulin B-chain, which are not observed in the crystal structure. While this conformational difference may result simply from the removal of solvent and addition of excess protons by the ESI, we propose that the migration may be enhanced by charge reduction during the ECD process itself because ion-dipole interactions are key to CB7 binding. The results of this study caution against using ECD-MS as a stand-alone structural probe for the determination of solution-phase binding sites.

Laser-induced fluorescence lifetimes are reported for several mass-selected gas-phase rhodamine cations, which were generated using electrospray ionization. A recently developed instrument consisting of a quadruple ion trap mass spectrometer coupled to a tunable laser excitation source is used for ion storage and irradiation, respectively. Time-resolved fluorescence measurements are carried out using time-correlated single photon counting (TCSPC), with a Single Photon Avalanche Diode (SPAD) for efficient detection of the light. The time-resolved data are well fit using single exponential decays. Measured lifetimes are 5.65 ± 0.04 ns for gaseous cationic rhodamine 575 (also known as rhodamine 19), 5.83 ± 0.06 ns for rhodamine 590, 5.87 ± 0.05 ns for rhodamine 6G and 6.92 ± 0.06 ns for rhodamine 640 (also known as rhodamine 101). These values are the first reports of the lifetimes of gaseous cationic rhodamine 575, 590 and 6G, while the uncertainty for the reported lifetime of 640 is an order of magnitude smaller than the previously reported value. The reported gas-phase fluorescence lifetimes for these rhodamines are all longer than their measured lifetimes in solution. Photo-physical processes leading to the increase in the fluorescence lifetimes of molecules in the gas phase compared to their solution behavior are discussed. Knowledge of the intrinsic lifetimes of rhodamines, as provided in this work, will aid the development of fluorescence resonance energy transfer as a structural probe of gaseous ions. It will also form a basis from which to better understand how interactions with solvent influence the properties of fluorescent dyes.

The structural characterization of gaseous biomolecular ions remains a challenging task. Here, we employ a combination of gas-phase hydrogen-deuterium exchange (HDX) and electron capture dissociation (ECD) mass spectrometry for gaining insights into the properties of two electrosprayed peptides: RA(9)K and RG(9)K. Mass analysis of ECD fragments provides spatially resolved labeling information. ND(3)-mediated HDX at peptide termini and amino acid side chains goes to completion within 1 s. Backbone amide labeling occurs more slowly, and proceeds in a structurally sensitive fashion. HDX is more extensive for RG(9)K than for RA(9)K, suggesting a more "open" conformation for the former. Residues 7-10 in RA(9)K are strongly protected, which indicates the presence of stable backbone hydrogen bonds at these sites. Our findings are consistent with the results of previous ion mobility measurements and computational investigations. Overall, it appears that the combination of gas-phase HDX and ECD represents a viable approach for uncovering structural features of biomolecular ions in the gas phase.

Although green fluorescent protein (GFP) is widely used in the biological sciences, the photophysics controlling GFP fluorescence versus non-radiative decay pathways are not well understood. In previous work (Forbes and Jockusch, J. Am. Chem. Soc. 2009), we reported that a gaseous anionic model chromophore of GFP, p -hydroxybenzylidene-2,3-dimethylimidazolone (HBDI − ), deactivates via electron photodetachment ( ePD ) as well as photofragmentation. Distinct electronic action spectra were measured for these two pathways upon activation of HBDI − in a quadrupole ion trap (QIT) mass spectrometer. Here, we explore the mechanisms of HBDI − dissociation following photoexcitation at two characteristic wavelengths: 410 nm, at which the dominant pathway is ePD and at 480 nm, at which the branching ratio between ePD and fragmentation depends strongly on the experimental conditions employed. The results indicate that ePD from HBDI − at 410 nm is a single photon process with a rate that is unaffected by changing the pressure of the helium bath gas. This is consistent with a prompt electron detachment process at 410 nm. At 480 nm, photofragmentation in the QIT requires absorption of more than one photon and is suppressed by collisions, while ePD results from both single and multiple photon absorption. A model that accounts for all these observations is discussed. ePD and effective absorption cross sections are estimated as is the rate of collisional cooling within the QIT. This work explores the connection between absorption and action spectroscopy as applied to the photophysics of a biologically important chromophore.

Studying the photophysical properties of molecules in the gasphase can be advantageous, because it reduces the complexity of the system examined by eliminating effects due to theinteractions between the molecule of interest and other species present in the local environment, including those with the solvent itself. Here, we report on theintrinsic properties of gaseous protonated rhodamine B (RBH+), a well-known xanthene-based dye. Protonated rhodamine B was transferred into the gas phase using electrospray ionization (ESI) and isolated in a quadrupole ion trap (QIT) mass spectrometer, which has been modified to enable laser-induced fluorescence spectroscopy of trapped ions. The gas-phase fluorescence excitation and emission spectra of RBH+show maxima (λex (max) = 531 nm and λem (max) = 542 nm, respectively)that lie at higher energy than those of RBH+ in solution. The fluorescence lifetime of gaseous RBH+ is 5.97 ± 0.12 ns, which is significantly longer than that of solution-phase rhodamine B. Gaseous rhodamine B is signficiantly brighter than monoethylamino rhodamines such as gaseous rhodamine 6G. Knowledge of the intrinsic photophysical properties of chromophores, such as those presented here for rhodamine B, will enable a better understanding of how the local environment of the chromophore modulates its properties.

The gas-phase fluorescence excitation, emission and photodissociation characteristics of three xanthene dyes (rhodamine 575, rhodamine 590, and rhodamine 6G) have been investigated in a quadrupole ion trap mass spectrometer. Measured gas-phase excitation and dispersed emission spectra are compared with solution-phase spectra and computations. The excitation and emission maxima for all three protonated dyes lie at higher energy in the gas phase than in solution. The measured Stokes shifts are significantly smaller for the isolated gaseous ions than the solvated ions. Laser power-dependence measurements indicate that absorption of multiple photons is required for photodissociation. Redshifts and broadening of the dispersed fluorescence spectra at high excitation laser power provide evidence of gradual heating of the ion population, pointing to a mechanism of sequential multiple-photon activation through absorption/emission cycling. The relative brightness in the gas phase follows the order R575(1.00)

The gas-phase structures of protonated and alkali-metal-cationized lysine (Lys) and epsilon-N-methyllysine (Lys(Me)) are investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser, in conjunction with ab initio calculations. IRMPD spectra of Lys.Li(+) and Lys.Na(+) are similar, but the spectrum for Lys.K(+) is different, indicating that the structure of lysine in these complexes depends on the metal ion size. The carbonyl stretch of a carboxylic acid group is clearly observed in each of these spectra, indicating that lysine is nonzwitterionic in these complexes. A detailed comparison of these spectra to those calculated for candidate low-energy structures indicates that the bonding motif for the metal ion changes from tricoordinated for Li and Na to dicoordinated for K, clearly revealing the increased importance of hydrogen-bonding relative to metal ion solvation with increasing metal ion size. Spectra for Lys(Me).M(+) show that Lys(Me), an analogue of lysine whose side chain contains a secondary amine, is nonzwitterionic with Li and zwitterionic with K and both forms are present for Na. The proton affinity of Lys(Me) is 16 kJ/mol higher than that of Lys; the higher proton affinity of a secondary amine can result in its preferential protonation and stabilization of the zwitterionic form.

Combining the selectivity of mass spectrometry (MS) with laser-induced fluorescence (LIF) presents a promising route to probe the intrinsic conformation, stability and dynamics of biological macromolecules. However, applications to proteins are in their infancy. Recent advances include the realization of Forster (fluorescence) resonance energy transfer (FRET) to provide nm-range distance constraints in de-solvated proteins, and measurement of dynamic fluorescence quenching rates to assess shorter-range interactions in peptides and Trp-cage. Temperature-dependent experiments employing FRET and dynamic quenching as conformational probes enable determination of enthalpy and entropy of conformational change in de-solvated biomolecules. These developments show the feasibility of using MS-LIF to dissect complex molecular interactions. For example, MS-LIF of protein-ligand complexes and partially hydrated proteins will better elucidate the energetics of specific binding interactions and the role of the solvent in protein structure and folding.

The intrinsic conformer specific vibrational spectra of two important subunits of the core pentasaccharide of N-linked glycans, the alpha(1,3) and alpha(1,6) dimannosides, have been recorded in the gas phase. Coupling these measurements with a computational exploration of their conformational landscapes has enabled their conformational assignment and has identified characteristic vibrational signatures associated with particular conformational families-including those that do or do not display inter-ring hydrogen bonding across the glycosidic linkage. In addition, the IR spectra of the monosaccharide moieties provide benchmarks, through which the conformational assignments can be refined. This introduces a general concept of modularity and secondary structure in oligosaccharides--essential for the success of similar studies on larger oligosaccharides in the future.

The conformation of phenyl-substituted monosaccharides (mannose, galactose, and glucose) and their singly hydrated complexes has been investigated in the gas phase by means of a combination of mass selected, conformer specific ultraviolet and infrared double resonance hole burning spectroscopy experiments, and ab initio quantum chemistry calculations. In each case, the water molecule inserts into the carbohydrate at a position where it can replace a weak intramolecular interaction by two stronger intermolecular hydrogen bonds. The insertion can produce significant changes in the conformational preferences of the carbohydrates, and there is a clear preference for structures where cooperative effects enhance the stability of the monosaccharide conformers to which the water molecule chooses to bind. The conclusions drawn from the study of monosaccharide-water complexes are extended to the disaccharide lactose and discussed in the light of the underlying mechanisms that may be involved in the binding of carbohydrate assemblies to proteins and the involvement, or not, of key structural water molecules.

The functional importance of carbohydrates in biological processes, particularly those involving specific molecular recognition, is immense. Characterizing the three-dimensional structures of carbohydrates and glycoconjugates and their interactions with other molecules, particularly the ubiquitous solvent, water, are key starting points on the road towards the understanding of these processes. The review introduces a new strategy, combining electronic and vibrational spectroscopy of mass-selected carbohydrate molecules and their hydrated (and also protonated) complexes, conducted under molecular beam conditions, with ab initio computation. Its early successes have revealed a uniquely powerful means of characterizing carbohydrate conformations and hydrated structures, the hydrogen-bonded networks they support (or which support them) and the specificity of their interactions with other molecules. The new information, obtained in the gas phase, complements that provided by more ‘traditional’ condensed phase ...

The structural investigation of a hydrated monosaccharide, phenyl β-d-glucopyranoside (PheβGlc), in the gas phase is presented. It is based upon ab initio computation coupled with the analysis of the resonant 2-photon ionization and IR ion-dip spectra of the singly hydrated clusters stabilized in a free jet expansion and follows an earlier investigation of the structure and conformations of the unhydrated sugar.1 Despite the potentially large set of possible binding sites and conformations, only two singly hydrated complexes are formed in the free jet expansion. Tentative structural assignments are made on the basis of comparisons with those already established for related systems; comparisons between the observed O−H vibrational frequencies and those computed for structures optimized at the B3LYP/6-31+G(d) level of theory, and in light of the relative energies of these structures, calculated at the single-point MP2/6-311+G(d,p) level are made. A discrepancy between the latest revision of the Gaussian 98 ...

The gas-phase conformation of the model glycoside, phenyl β-D-galactopyranoside (phe-β-D-gal) was examined using a combination of resonant two-photon ionization (R2PI) and resonant ion-dip infrared spectroscopy (RIDIRS) in tandem with electronic structure theory calculations. A single conformer, in which the hydroxy methyl is in a gauche+ orientation, is predominant in the free-jet expansion. This conformer is the analogue of the lowest-energy conformer found in a previous study of phenyl β-D-glucopyranoside. A minor second conformer has also been identified. Other weak bands in the R2PI spectrum have been attributed to hot bands though it is possible that other conformers are also present at low abundance. The dominant conformer identified in this work has the same (gauche+) orientation of the hydroxy methyl group as the major rotamer identified in solution by NMR spectroscopy.

An improved model for the slow infrared laser photodissociation of large biomolecules is proposed. As has been previously shown by master equation and Monte Carlo calculations, large, laser-heated ...

The mode of metal ion and water binding to the amino acid valine is investigated using both theory and experiment. Computations indicate that without water, the structure of valine is nonzwitterionic. Both Li(+) and Na(+) are coordinated to the nitrogen and carbonyl oxygen (NO coordination), whereas K(+) coordinates to both oxygens (OO coordination) of nonzwitterionic valine. The addition of a single water molecule does not significantly affect the relative energies calculated for the cationized valine clusters. Experimentally, the rates of water evaporation from clusters of Val.M(+)(H(2)O)(1), M = Li, Na, and K, are measured using blackbody infrared radiative dissociation. The dissociation rate from the valine complex is compared to water evaporation rates from model complexes of known structure. These results indicate that the metal ion in the lithiated and the sodiated clusters is NO-coordinated to nonzwitterionic valine, while that in the potassiated cluster has OO coordination, in full agreement with theory. The zwitterionic vs nonzwitterionic character of valine in the potassiated cluster cannot be distinguished experimentally. Extensive modeling provides strong support for the validity of inferring structural information from the kinetic data.

The evaporation of water from hydrated alkaline earth metal ions, produced by electrospray ionization, was studied in a Fourier transform mass spectrometer. Zero-pressure-limit dissociation rate constants for loss of a single water molecule from the hydrated divalent metal ions, M2+(H2O)n (M = Mg, Ca, and Sr for n = 5–7, and M = Ba for n = 4–7), are measured as a function of temperature using blackbody infrared radiative dissociation. From these values, zero-pressure-limit Arrhenius parameters are obtained. By modeling the dissociation kinetics using a master equation formalism, threshold dissociation energies (Eo) are determined. These reactions should have a negligible reverse activation barrier; therefore, Eo values should be approximately equal to the binding energy or hydration enthalpy at 0 K. For the hepta- and hexahydrated ions at low temperature, binding energies follow the trend expected on the basis of ionic radii: Mg > Ca > Sr > Ba. For the hexahydrated ions at high temperature, binding energies follow the order Ca > Mg > Sr > Ba. The same order is observed for the pentahydrated ions. Collisional dissociation experiments on the tetrahydrated species result in relative dissociation rates that directly correlate with the size of the metals. These results indicate the presence of two isomers for hexahydrated magnesium ions: a low-temperature isomer in which the six water molecules are located in the first solvation shell, and a high-temperature isomer with the most likely structure corresponding to four water molecules in the inner shell and two water molecules in the second shell. These results also indicate that the pentahydrated magnesium ions have a structure with four water molecules in the first solvation shell and one in the outer shell. The dissociation kinetics for the hexa- and pentahydrated clusters of Ca2+, Sr2+, and Ba2+ are consistent with structures in which all the water molecules are located in the first solvation shell.

The dissociation kinetics of protonated n-acetyl-L-alanine methyl ester dimer (AcAlaME(d)), imidazole dimer, and their cross dimer were measured using blackbody infrared radiative dissociation (BIRD). Master equation modeling of these data was used to extract threshold dissociation energies (E(o)) for the dimers. Values of 1.18 +/- 0.06, 1.11 +/- 0.04, and 1.12 +/- 0.08 eV were obtained for AcAlaME(d), imidazole dimer, and the cross dimer, respectively. Assuming that the reverse activation barrier for dissociation of the ion-molecule complex is negligible, the value of E(o) can be compared to the dissociation enthalpy (DeltaH(d) degrees ) from HPMS data. The E(o) values obtained for the imidazole dimer and the cross dimer are in agreement with HPMS values; the value for AcAlaME(d) is somewhat lower. Radiative rate constants used in the master equation modeling were determined using transition dipole moments calculated at the semiempirical (AM1) level for all dimers and compared to ab initio (RHF/3-21G*) calculations where possible. To reproduce the experimentally measured dissociation rates using master equation modeling, it was necessary to multiply semiempirical transition dipole moments by a factor between 2 and 3. Values for transition dipole moments from the ab initio calculations could be used for two of the dimers but appear to be too low for AcAlaME(d). These results demonstrate that BIRD, in combination with master equation modeling, can be used to determine threshold dissociation energies for intermediate size ions that are in neither the truncated Boltzmann nor the rapid energy exchange limit.

Blackbody infrared radiative dissociation (BIRD) spectra of singly and doubly protonated bradykinin and its analogues are measured in a Fourier-transform mass spectrometer. Rate constants for dissociation are measured as a function of temperature with reaction delays up to 600 s. From these data, Arrhenius activation parameters in the zero-pressure limit are obtained. The activation parameters and dissociation products for the singly protonated ions are highly sensitive to small changes in ion structure. The Arrhenius activation energy (E(a)) and pre-exponential (or frequency factor, A) of the singly protonated ions investigated here range from 0.6 to 1.4 eV and 10(5) to 10(12) s(-1), respectively. For bradykinin and its analogues differing by modification of the residues between the two arginine groups on either end of the molecule, the singly and doubly protonated ions have average activation energies of 1.2 and 0.8 eV, respectively, and average A values of 10(8) and 10(12) s(-1), respectively, i.e., the presence of a second charge reduces the activation energy by 0.4 eV and decreases the A value by a factor of 10(4). This demonstrates that the presence of a second charge can dramatically influence the dissociation dynamics of these ions. The doubly protonated methyl ester of bradykinin has an E(a) of 0.82 eV, comparable to the value of 0.84 eV for bradykinin itself. However, this value is 0.21 +/- 0.08 eV greater than that of singly protonated methyl ester of bradykinin, indicating that the Coulomb repulsion is not the most significant factor in the activation energy of this ion. Both singly and doubly protonated Lys-bradykinin ions have higher activation energies than the corresponding bradykinin ions indicating that the addition of a basic residue stabilizes these ions with respect to dissociation. Methylation of the carboxylic acid group of the C-terminus reduces the E(a) of bradykinin from 1.3 to 0.6 eV and the A factor from 1012 to 105 s(-1). This modification also dramatically changes the dissociation products. Similar results are observed for [Ala(6)]-bradykinin and its methyl ester. These results, in combination with others presented here, provide experimental evidence that the most stable form of singly protonated bradykinin is a salt-bridge structure.

2',7'-Dichloro- and 2',7'-difluorofluoresceins are superior alternatives to underivatized fluorescein. Although several studies characterizing their condensed-phase photophysical properties have been reported, little is known about their intrinsic characteristics. Here, the gas-phase properties of three charge states of each fluorescein are characterized using a quadrupole ion trap mass spectrometer which has been modified for spectroscopy. Electronic action spectra, constructed by monitoring the extent of photodissociation as a function of excitation wavelength, indicate that the gaseous dianions and cations resemble their solution-phase counterparts. In contrast, a large shift in the electronic action spectra of the monoanions indicates the presence of a different tautomer in the gas phase than that present in solution. The gaseous monoanion is deprotonated on the xanthene ring, rather than being deprotonated on the pendant group as found in soluion. The dianions and cations do not emit detectable fluorescence in the gas phase. In contrast, the monoanions do fluoresce, but the emission intensity is low and the spectra are broad. This work illustrates the effect of halogenation on the intrinsic properties of the dyes and provides useful fundamental understanding that promises to aid the development more robust fluorescent dyes.

Fluorescein (FL) and its derivative 2′,7′-dichlorofluoroescein (DCF) are well-known fluorescent dyes used in many biological and biochemical applications. Although extensive studies have been carried out to investigate their chemical and photophysical properties in different solvent media, little is known about their intrinsic behaviors in the gas phase. Here, infrared multiple photon dissociation (IRMPD) action spectra are reported for the three charged prototropic forms of FL and DCF and compared with computed IR spectra from electronic structure calculations. In each case, the measured spectra show good agreement with the calculated spectra of the lowest energy computed conformer. Moreover, the major bands of the monoanion IRMPD spectra show striking similarities to those of the dianions and are quite different from those of the cations. These experimental results clearly indicate that the gaseous monoanions are predominantly deprotonated on the xanthene chromophore, rather than the benzoate deprotonation site favored in solution. Investigations such as this, which provide a better understanding of intrinsic properties of ionic dyes, forms a baseline from which to elucidate solvent effects and will aid the rational design of dyes possessing desirable fluorescence properties.

Despite the many successes of mass spectrometry in the analysis of biological samples, the need to better understand the correlation between condensed-phase properties and those of electrospray species remains. In particular, the link between structures in the condensed phase and in the gaseous environment of the mass spectrometer is still elusive. Here, we show that fluorescence resonance energy transfer (FRET) can be used to probe the conformations of gaseous biopolymers which are formed by electrospray ionization (ESI) and manipulated in a quadrupole ion trap mass spectrometer. A rhodamine dye pair suitable for gas-phase FRET is characterized. Both steady state spectra and lifetime measurements are used to monitor energy transfer in a series of dye-labeled polyproline-based peptides. FRET efficiency is explored as a function of peptide chain length and charge state. For the peptide with eight proline repeats, virtually complete energy transfer is observed. For the peptide with 14 proline repeats, energy transfer decreases as the charge state increases, consistent with Coulomb repulsion induced elongation of the peptide backbone. FRET measurements of the longest peptide examined, which has 20 proline repeats, indicates that the peptide adopts a bent configuration. Evidence for multiple conformations present within the ensemble of trapped ions is provided by fluorescence lifetime measurements. Gas-phase FRET measurements promise to be a new route to probe the conformations of large gaseous ions.

The structures of cationized arginine complexes [Arg + M]+, (M = H, Li, Na, K, Rb, Cs, and Ag) and protonated arginine methyl ester [ArgOMe + H]+ have been investigated in the gas phase using calculations and infrared multiple-photon dissociation spectroscopy between 800 and 1900 cm-1 in a Fourier transform ion cyclotron resonance mass spectrometer. The structure of arginine in these complexes depends on the identity of the cation, adopting either a zwitterionic form (in salt-bridge complexes) or a non-zwitterionic form (in charge-solvated complexes). A diagnostic band above 1700 cm-1, assigned to the carbonyl stretch, is observed for [ArgOMe + H]+ and [Arg + M]+, (M = H, Li, and Ag), clearly indicating that Arg in these complexes is non-zwitterionic. In contrast, for the larger alkali-metal cations (K+, Rb+, and Cs+) the measured IR-action spectra indicate that arginine is a zwitterion in these complexes. The measured spectrum for [Arg + Na]+ indicates that it exists predominantly as a salt bridge with zwitterionic Arg; however, a small contribution from a second conformer (most likely a charge-solvated conformer) is also observed. While the silver cation lies between Li+ and Na+ in metal-ligand bond distance, it binds as strongly or even more strongly to oxygen-containing and nitrogen-containing ligands than the smaller Li+. The measured IR-action spectrum of [Arg + Ag]+ clearly indicates only the existence of non-zwitterionic Arg, demonstrating the importance of binding energy in conformational selection. The conformational landscapes of the Arg-cation species have been extensively investigated using a combination of conformational searching and electronic structure theory calculations [MP2/6-311++G(2d,2p)//B3LYP/6-31+G(d,p)]. Computed conformations indicate that Ag+ is di-coordinated to Arg, with the Ag+ chelated by both the N-terminal nitrogen and Neta of the side chain but lacks the strong M+-carbonyl oxygen interaction that is present in the tri-coordinate Li+ and Na+ charge-solvation complexes. Experiment and theory show good agreement; for each ion species investigated, the global-minimum conformer provides a very good match to the measured IR-action spectrum.

Background By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to e stablish s ilent c hromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions. Results Targeted silencing by Rtt107/Esc4 was dependent on the SIR genes, which encode obligatory structural and enzymatic components of yeast silent chromatin. Based on its sequence, Rtt107/Esc4 was predicted to contain six BRCT motifs. This motif, originally identified in the human breast tumor suppressor gene BRCA1, is a protein interaction domain. The targeted silencing activity of Rtt107/Esc4 resided within the C-terminal two BRCT motifs, and this region of the protein bound to Sir3 in two-hybrid tests. Deletion of RTT107/ESC4 caused sensitivity to the DNA damaging agent MMS as well as to hydroxyurea. A two-hybrid screen showed that the N-terminal BRCT motifs of Rtt107/Esc4 bound to Slx4, a protein previously shown to be involved in DNA repair and required for viability in a strain lacking the DNA helicase Sgs1. Like SLX genes, RTT107ESC4 interacted genetically with SGS1; esc4 Δ sgs1 Δ mutants were viable, but exhibited a slow-growth phenotype and also a synergistic DNA repair defect. Conclusion Rtt107/Esc4 binds to the silencing protein Sir3 and the DNA repair protein Slx4 via different BRCT motifs, thus providing a bridge linking silent chromatin to DNA repair enzymes.

The gas-phase structures of cationized arginine, Arg.M(+), M = Li, Na, K, Rb, and Cs, were studied both by hybrid method density functional theory calculations and experimentally using low-energy collisionally activated and thermal radiative dissociation. Calculations at the B3LYP/LACVP++** level of theory show that the salt-bridge structures in which the arginine is a zwitterion (protonated side chain, deprotonated C-terminus) become more stable than the charge-solvated structures with increasing metal ion size. The difference in energy between the most stable charge-solvated structure and salt-bridge structure of Arg.M(+) increases from -0.7 kcal/mol for Arg.Li(+) to +3.3 kcal/mol for Arg.Cs(+). The stabilities of the salt-bridge and charge-solvated structures reverse between M = Li and Na. These calculations are in good agreement with the results of dissociation experiments. The low-energy dissociation pathways depend on the cation size. Arginine complexed with small cations (Li and Na) loses H(2)O, while arginine complexed with larger cations (K, Rb, and Cs) loses NH(3). Loss of H(2)O must come from a charge-solvated ion, whereas the loss of NH(3) can come from the protonated side chain of a salt-bridge structure. The results of dissociation experiments using several cationized arginine derivatives are consistent with the existence of these two distinct structures. In particular, arginine methyl esters, which cannot form salt bridges, dissociate by loss of methanol, analogous to loss of H(2)O from Arg.M(+); no loss of NH(3) is observed. Although dissociation experiments probe gas-phase structure indirectly, the observed fragmentation pathways are in good agreement with the calculated lowest energy isomers. The combination of the results from experiment and theory provides strong evidence that the structure of arginine-alkali metal ion complexes in the gas phase changes from a charge-solvated structure to a salt-bridge structure as the size of the metal ion increases.

Metal ions play an important role in the function of many metalloenzymes. For example, the interactions of regulatory proteins such as calmodulin with other proteins depend on whether Ca2+ is specifically bound.1 Studies of metal ion hydration provide information not only about the metal ion chemistry in solution, but can lead to an improved understanding of the structure and function of many biomolecules in which metal ion interactions play a role. The hydration enthalpies of singly charged metal ions in the gas phase have been extensively investigated by using a variety of experimental techniques.2 In contrast, gas-phase hydration studies of di- and trivalent metal ions are significantly more limited.3–5 Kebarle and co-workers measured free energies of hydration for a series of divalent metal ions bound to between 7 and 13 water molecules.4b,d From these values, hydration enthalpies for water molecules in the second solvation shell were estimated. Recently, calculations of successive hydration enthalpies of both inner and outer solvent shell water molecules for several divalent metal ions have been reported.6,7 Here, binding energies of water molecules to both divalent calcium and nickel ions are measured by using blackbody infrared radiative dissociation (BIRD) and master equation modeling. These values are considerably lower than MP2 hydration enthalpies reported previously.6 They are in better agreement with recently reported B3LYP values calculated with large basis sets.7 To our knowledge, these are the first experimental measurements of the binding energy of individual inner shell water molecules to divalent metal ions. Experimental measurements are performed with an external electrospray ionization source Fourier transform mass spectrometer. This instrument and the BIRD experiments have been described previously.8 Hydrated metal ions are generated from ~10−4 M chloride salt solutions with nanoelectrospray ionization. The hydrated ions are trapped and thermalized by using a pulse of N2 gas (10−6 Torr). The ion of interest is mass selected and dissociated for times ranging from 10 to 500 s. At the low pressures during the reaction times (

Photodissociation kinetics of the protonated pentapeptide leucine enkephalin measured using a cw CO(2) laser and a Fourier-transform mass spectrometer are reported. A short induction period, corresponding to the time required to raise the internal energy of the ion population to a (dissociating) steady state, is observed. After this induction period, the dissociation data are accurately fit by first-order kinetics. A plot of the log of the unimolecular dissociation rate constant, k(uni), as a function of the log of laser power is linear at low laser powers (9 W, k(uni)0.05 s(-1)), but tapers off at high laser power (9-33 W, k(uni) = 0.05-7 s(-1)). The entire measured dissociation curve can be accurately fit by an exponential function plus a constant. The experiment is simulated using a master equation formalism. In the model, the laser radiation is described as an energetically flat-topped distribution which is spatially uniform. This description is consistent with experimental results which indicate that ion motion within the cell averages out spatial inhomogeneities in the laser light. The model has several adjustable parameters. The effect of varying these parameters on the calculated kinetics and power dependence curves is discussed. A procedure for determining a limited range of threshold dissociation energy, E(o), which fits both the measured induction period and power dependence curves, is presented. Using this procedure, E(o) of leucine enkephalin is determined to be 1.12-1.46 eV. This result is consistent with, although less precise than, values measured previously using blackbody infrared radiative dissociation. Although the blackbody dissociation results were used as a starting point to search for fits of the master equation model to experiment, these results demonstrate that it is, in principle, possible to determine a limited range of E(o) from slow infrared multiphoton dissociation data alone.

The beta(1--4) glycosidic linkage found in lactose is a prevalent structural motif in many carbohydrates and glycoconjugates. Using UV and IR ion-dip spectroscopies to probe benzyl lactoside isolated in the gas phase, we find that the disaccharide unit adopts only a single, rigid structure. Its fully resolved infrared ion-dip spectrum is in excellent agreement with that of the global minimum structure computed ab initio. This has glycosidic torsion angles of phi(H) (H1-C1-O-C4') approximately 180 degrees and psi(H) (C1-O-C4'-H4') approximately 0 degrees which correspond to a rotation of approximately 150 degrees about the glycosidic bond compared to the accepted solution-phase conformation. We discuss the biological implications of this discovery and the generality of the strategies employed in making it.

A novel potassium channel antagonist has been purified from the defensive mucus secreted by Calliostoma canaliculatum, a marine snail found in the temperate coastal waters of the western Pacific. The toxin is expelled from the hypobranchial gland as part of a defensive response and is contained within a viscous matrix that minimizes dilution and degradation. The active compound was isolated by multistage microbore HPLC separations followed by bioactivity assays. Nuclear magnetic resonance, combined with electrospray ionization Fourier-transform ion cyclotron resonance and electrospray ionization ion trap mass spectrometry indicate that the active component is a heretofore unknown indole-derivative, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT). Exudates from the hypobranchial glands of various marine mollusks have been sources for dye compounds such as 6–6 dibromoindigo, the ancient dye Tyrian purple. BrMT represents the first correlation of a hypobranchial gland exudate with a molecular response. Voltage clamp experiments with a number of K channel subtypes indicate that BrMT inhibits certain voltage-gated K channels of the Kv1 subfamily.

The mechanism of fluorescence and fluorescence quenching of the green fluorescent protein (GFP) is not well-understood. To gain insight into the effect of the surrounding protein on the chromophore buried at its center, the intrinsic electronic absorption and deactivation pathways of a gaseous model chromophore, p-hydroxybenzylidene-2,3-dimethylimidazolone (HBDI) were investigated. No fluorescence from photoactivated gaseous HBDI(-) was detected in the range 480-1100 nm, in line with the ultrafast rate of internal conversion of HBDI(-) in solution. Two different gas-phase deactivation pathways were found: photofragmentation and electron photodetachment. Electronic action spectra for each deactivation pathway were constructed by monitoring the disappearance of HBDI(-) and appearance of product ions as a function of excitation wavelength. The action spectra measured for each pathway are distinct, with electron photodetachment being strongly favored at higher photon energies. The combined (total) gas-phase action spectrum has a band origin at 482.5 nm (23340 cm(-1)) and covers a broad spectral range, 390-510 nm. This extended gas-phase action spectrum exhibits vibronic activity that matches well with the results of previous cold condensed-phase experiments and high-level in vacuo computations, with features evident at +550, +1500, and +2800 cm(-1) with respect to the band origin.

The dissociation kinetics of proton-bound dimers of betaine with molecules of comparable gas-phase basicity were investigated using blackbody infrared radiative dissociation (BIRD). Threshold dissociation energies were obtained from these data using master equation modeling. For bases that have comparable or higher gas-phase basicity, the binding energy of the protonated base.betaine complex is approximately 1.4 eV. For molecules that are approximately 2 kcal/mol or more less basic, the dissociation energy of the complexes is approximately 1.2 eV. The higher binding energy of the former is attributed to an ion-zwitterion structure which has a much larger ion-dipole interaction. The lower binding energy for molecules that are approximately 2 kcal/mol or more less basic indicates that an ion-molecule structure is more favored. Semiempirical calculations at both the AM1 and PM3 levels indicate the most stable ion-molecule structure is one in which the base interacts with the charged quaternary ammonium end of betaine. These results indicate that the measurement of binding energies of neutral molecules to biological ions could provide a useful probe for the presence of zwitterions and salt bridges in the gas phase. From the BIRD data, the gas-phase basicity of betaine obtained from the kinetic method is found to be 239.2 +/- 1.0 kcal/mol. This value is in excellent agreement with the value of 239.3 kcal/mol (298 K) from ab initio calculations at the MP2/6-31+g** level. The measured value is slightly higher than those reported previously. This difference is attributed to entropy effects. The lower ion internal energy and longer time frame of BIRD experiments should provide values closer to those at standard temperature.

Blackbody infrared radiative dissociation spectra of the (M + 5H)5+ through (M + 11H)11+ ions of the protein ubiquitin (8.6 kDa) formed by electrospray ionization were measured in a Fourier-transform mass spectrometer. The 5+ ion dissociates exclusively by loss of water and/or ammonia, whereas the 11+ charge state dissociates only by formation of complementary y and b ions. These two processes are competitive for intermediate charge state ions, with the formation of y and b ions increasingly favored for the higher charge states. The y and b ions are formed by cleavage of the backbone amide bond on the C-terminal side of acidic residues exclusively, with cleavage adjacent to aspartic acid favored. Thermal unimolecular dissociation rate constants for the dissociation of each of these charge states were measured. From the temperature dependence of these rates, Arrhenius activation parameters in the rapid energy exchange limit are obtained. The activation energies (Ea) and preexponential factors (A) for the 5+, 8+, and 9+ ions are 1.2 eV and 10(12) s-1, respectively. These values for the 6+ and 7+ ions are 0.9-1.0 eV and 10(9) s-1, and those for the 10+ and 11+ ions are 1.6 eV and 10(16)-10(17) s-1. Thus, with the exception of the 5+ ion, the higher charge states of ubiquitin have larger dissociation activation energies than the lower charge states. The different A factors observed for production of y and b ions from different precursor charge states indicate that they are formed by different mechanisms, ranging from relatively complex rearrangements to direct bond cleavages. These results clearly demonstrate that the relative dissociation rates of large biomolecule ions by themselves are not necessarily a reliable indicator of their relative dissociation energies, even when similar fragment ions are formed.

A flexible interface to perform optical spectroscopic measurements on gaseous ions stored in a modified commercial quadrupole ion trap (QIT) mass spectrometer is described. The modifications made to the mass spectrometer did not adversely affect its operating characteristics. Gas-phase ions are produced using electrospray ionization, mass isolated and stored in the trapping mass spectrometer. The ions are subsequently irradiated with visible light from a tunable laser and dispersed fluorescence spectra are recorded simultaneously. Mass spectra are recorded after the irradiation period. This set-up allows us to track a range of possible outcomes upon photoexcitation of selected ions including fluorescence, photofragmentation and photodetachment of electrons. The experimental set-up is characterized using rhodamine 590, which is a methyl ester variant of rhodamine 6G. Fluorescence excitation and emission spectra of gaseous rhodamine 590 are measured and compared with solution-phase spectra. Excitation and emission maxima for the gaseous ions are found to lie at higher energy than for the solvated rhodamine 590. In addition, the gas-phase Stokes shift is significantly smaller than the solution-phase Stokes shift. The effects of several experimental parameters on the observed fluorescence signal are investigated, including laser power, relative number of ions, q(z) trapping parameter and buffer gas pressure. In addition to its use for the photophysical characterization of the intrinsic properties of ionic chromophores, this set-up may be used to investigate the properties of mass-selected, dye-labeled biomolecules, both alone and in well-defined complexes and clusters.